The lymphoma-associated NPM-ALK oncogene elicits a p16INK4a/pRb-dependent tumor-suppressive pathway by Paola Martinelli, Paola Bonetti, Cristina Sironi, Giancarlo Pruneri, Caterina Fumagalli, Paola Rafaniello Raviele, Sara Volorio, Stefano Pileri, Roberto Chiarle, Fiona Kate Elizabeth McDuff, Betsabeh Khoramian Tusi, Suzanne D. Turner, Giorgio Inghirami, Pier Giuseppe Pelicci, and Emanuela Colombo Blood Volume 117(24):6617-6626 June 16, 2011 ©2011 by American Society of Hematology

NPM-ALK inhibits p53 activity and it induces a senescent checkpoint in primary fibroblasts. NPM-ALK inhibits p53 activity and it induces a senescent checkpoint in primary fibroblasts. (A-F) WT MEFs were infected with control (Ctrl) and NPM-ALK (NA) or RASV12 expressing retroviruses. (A) Cells were analyzed (5 days after infection) by immunofluorescence (magnification ×600) and Western blot (B) with an anti-γH2AX antibody (representative micrographs and percentage of labeled cells are shown). (C) Growth curve of infected cells plated after infection and counted daily (cell numbers are expressed relative to day 1; *P < .05 relative to control cells infected with an empty vector). (D) β-galactosidase detection (representative micrographs [magnification ×600] and percentage of positive cells) and anti-BrdU staining (percentage of positive cells relative to control cells) performed 6 and 4 days after infection, respectively. (E) Western blotting of p53, ARF, p16INK4a, NPM-ALK, and tubulin expression, 4 days after infection. (F) Western blotting of pRb, NPM-ALK, cyclin A, and tubulin expression, 3 days after infection. (G) Luciferase assay in WT MEFs transduced with the pGL13 p53-reporter, the CMV-βGal vector, and increasing amounts of vectors expressing NPM-ALK or the K210R kinase-dead NPM-ALK mutant (or a control empty vector). Data are from 3 independent experiments and normalized to βGal. *P < .05 relative to control cells infected with an empty vector. (H) Same Luciferase assay as in panel G comparing a single dose (100 ng) of NPM-ALK– and RASV12-expressing vectors. Paola Martinelli et al. Blood 2011;117:6617-6626 ©2011 by American Society of Hematology

NPM-ALK induced senescence requires an intact p16INK4a/pRb pathway. NPM-ALK induced senescence requires an intact p16INK4a/pRb pathway. Growth curves of (A) p16INK4a−/− or (B) Rb1−/− MEFs infected with the indicated retroviruses. Values are expressed relative to day 1. In each panel, results are representative of at least 3 independent experiments. *P values are relative to control cells infected with empty vectors. (C) Methylcellulose-CFU of WT or p16INK4a−/− lin− cells infected with the indicated retroviruses (graph and representative images). Values are expressed relative to control infected cells. (D) Serial replating of WT and p16INK4a−/− lin− cells infected with the indicated retroviruses. CFUs are expressed relative to the first count for each sample. Results are representative of 3 experiments. *P < .05 relative to control cells infected with an empty vector. Paola Martinelli et al. Blood 2011;117:6617-6626 ©2011 by American Society of Hematology

NPM-ALK activates the p16INK4a pathway in vivo. NPM-ALK activates the p16INK4a pathway in vivo. (A) SA-β-gal staining of frozen sections from 1.5-month-old mice of the indicated genotypes (representative images [magnification ×400] and percentage of positive cells). (B) Percentage of Ki67-positive cells from thymocyte cell-suspensions of 1.5-month-old NPM-ALK transgenic or control littermates, as evaluated by FACS analysis. Results were obtained from 3 independent experiments, each performed using at least 2 mice of each genotype. (C) Survival curves of NPM-ALK transgenic mice of different genotypes for p16INK4a, as indicated. Median time to death is indicated in the inset. (D) Western blot analyses of p16INK4a, p53, and ARF expression in tumors derived from NPM-ALK transgenic mice of different p16INK4a genotypes, as indicated. Tubulin or vinculin was used as loading control. C+ indicates WT MEFs (positive control); C−, p16INK4a- or Tp53-null MEFs (negative controls). Paola Martinelli et al. Blood 2011;117:6617-6626 ©2011 by American Society of Hematology

Expression of p16INK4a, p53, and pRb in 19 tumor specimens from ALCL ALK+ patients by immunohistochemical analysis with specific antibodies. Expression of p16INK4a, p53, and pRb in 19 tumor specimens from ALCL ALK+ patients by immunohistochemical analysis with specific antibodies Paola Martinelli et al. Blood 2011;117:6617-6626 ©2011 by American Society of Hematology

p16INK4a reactivation limits growth of ALCL cell lines. p16INK4a reactivation limits growth of ALCL cell lines. (A) Western blotting analysis of pRb, p16INK4a, phosphorylated pRb (serine 807-811), NPM-ALK, and tubulin expression in 6 different ALCL cell lines (as indicated). (B) Left panel: Western blot analysis of NPM-ALK, p16INK4a, and tubulin expression at different times (as indicated) in SU-DHL-1 cells treated with doxocyclin to induce shRNA interference of NPM-ALK. Right panel: p16INK4a mRNA quantification by qPCR analysis with specific primers. Data were standardized with ribosomal RNA and normalized against control untreated cells. (C) Karpas299 and DEL cell lines were infected with control (Ctrl) and p16INK4a-expressing retroviruses. Left panel: at day 4, cells were collected and expression of p16INK4a, phosphoRb, and tubulin evaluated by Western blot. Right panels: infected cells were selected, plated, and counted daily. (D) Karpas299 and DEL cell lines were treated with 5-azacytidine (AZA) for 6 days. Left panel: at day 5, cells were collected and expression of p16INK4a, phosphoRb, and tubulin evaluated by Western blot. Right panels: cells were plated and counted daily. Paola Martinelli et al. Blood 2011;117:6617-6626 ©2011 by American Society of Hematology

NPM-ALK induces p16INK4a promoter de-methylation. NPM-ALK induces p16INK4a promoter de-methylation. (A) Top panel: chromatin immunoprecipitation analysis of p16INK4a promoter H3K27me3 levels in WT MEFs infected with the indicated retroviruses. Precipitated DNA was amplified by qPCR using primers located in the p16INK4a promoter region. Enrichments are presented as percentage of total input DNA. Bottom panel: Q-PCR analysis of p16INK4a mRNA levels in the same cells, expressed as fold increase with respect to the day 1 control vector. (B) Growth curves of WT MEFs infected with a control lentivirus or lentiviruses expressing JunB-specific shRNAs, in the presence or absence of NPM-ALK, as indicated. Values are expressed relative to day 1 for each sample. *P < .05 with respect to control cells infected with a control vector. (C) Western blotting of p16INK4a, JunB, and NPM-ALK expression in the same cell samples as in panel B, 4 days after infection. Tubulin was used as a loading control. p16INK4a protein levels were calculated by densimetric analysis and normalized to control (Ctrl.) and tubulin band intensities. (D) Western blotting of Jmjd3 and p16INK4a expression levels in WT MEFs infected with NPM-ALK or control viruses. p16INK4a protein levels were calculated as in panel C. (E) Growth curves of WT and Jmjd3 null MEFs infected as indicated. Values are expressed relative to day 1 for each sample. (F) Western blotting of p16INK4a and NPM-ALK expression levels in WT and Jmjd3-null MEFs in the same cell samples as in panel E, 4 days after infection. p16INK4a protein levels were calculated as in panel C. (G) Left panel: Western blot analysis of STAT3, p16INK4a, and tubulin expression at different times (as indicated) in TS cells treated with doxocyclin to induce shRNA interference targeting STAT3. Right panel: p16INK4a mRNA quantification by qPCR analysis with specific primers. Data were standardized with ribosomal RNA and normalized against control untreated cells. Paola Martinelli et al. Blood 2011;117:6617-6626 ©2011 by American Society of Hematology